The recent discovery that patient-specific induced pluripotent stem cells (iPS) can be generated from somatic cells using defined factors has provided unprecedented opportunities for generating unlimited sources of autologous, transplantable hematopoietic stem-progenitor cells (HSC). The clinical safety of stem cell-like iPS currently remains in question, however, since current iPS methodologies utilize integrating retroviruses expressing proto-oncogenes (e.g., Myc, Klf4, and SV40 T antigen), and thus produce cells with great potential for malignant transformation. Moreover, the efficient generation of adult, long-term engrafting HSC from pluripotent human embryonic stem cells (hESC) currently remains elusive. In this proposal, we assemble an interactive team of basic and translational scientists to address these major problems. To achieve the goal of safely generating and expanding iPS-HSC for treatment of hematologic disorders, we will focus on key developmental, transcriptional, and epigenetic mechanisms that orchestrate the expansion of de novo HSC from bone marrow (BM) stem cells, hESC, and iPS (derived from patient-specific fibroblasts, mesenchymal stem cells (MSC), CD34+ progenitors, and B-lymphoid cells). We will strive to replace ectopically-expressed defined factors with alternative reprogramming protocols that utilize non- integrating vectors, miRNAs, siRNAs, and chromatin-modifying small molecules. The goal of the following four Collaborative Projects is to generate clinically useful, transplantable iPS-HSC that lack malignant potential. Project I: Generation of HSC from hESC and iPS (PI: Zambidis). This project will: generate engraftable human HSC from iPS-derived hemangioblasts expressing CD143 by manipulating upstream signals (Notch), and regulatory factors (CDX-Hox);generate iPS from adult CD34+ HSC;investigate the role of miRNAs that regulate stem cell renewal. Project II. Specification and Expansion of Definitive HSC (PI: Friedman). This project will: investigate regulation of RUNX1 by upstream signals (Wnt, Notch, Hox) that mediate HSC emergence;use iPS generated from RUNX1(-/-) mice, ESC, and Runx1 variants to delineate mechanisms that mediate Runx1 specification of definitive HSC;investigate whether inhibition of HSC differentiation facilitates iPS-HSC formation/expansion using PU.1-/-, C/EBP1-/-, and GATA1-/- somatic cells. Project III. Mechanistic Role of c-Myc in iPS Generation (PI: Dang). This project will: delineate the role of a Myc-responsive miRNA cluster in induced pluripotency;generate iPS lacking using B cells at various stages of development. Project IV. Epigenetic Determinants of Neoplasia in iPS (PI: Baylin). This project will: examine the key shared aspect of both neoplasia and iPS reprogramming: abnormal epigenetic gene silencing;determine the degree to which this occurs via various iPS protocols. By drawing upon the broad clinical expertise at Johns Hopkins, our aim is to ultimately lead a translational program for safe iPS-HSC therapies, within the overall project time frame.